A Simple, Rapid, and Effective Heparinase Protocol to Enable Nucleic Acid Study from Frozen Heparinized Plasma

Cell-free RNAs (cfRNAs) are promising analytes as non-invasive biomarkers and have even greater potential if tied in with metabolomics. Plasma is an optimal source for cfRNAs but is often derived from a variety of anticoagulants. Plasma obtained in heparin is suitable for metabolomics but is difficult to utilize for qPCR-based downstream analysis. In the present study, we aimed to develop a simple, time-efficient, and cost-effective heparinase protocol, followed by library preparation and sequencing of human plasma cfRNAs drawn and stored in heparin at −80 °C for several years. Blood was collected in CPT™ sodium heparin tubes from patients with chronic HCV infection (NCT02400216) at the National Institutes of Health (NIH) Clinical Center. Plasma cfRNAs were treated with heparinase I and used for library preparation and next-generation sequencing (NGS). Heparinase treatment maintained RNA integrity and allowed for successful library preparation for all the study subjects even with 7 ng of cfRNAs as starting material. The classification report derived from Pavian R package v1.2.0 showed no artificial reads. The abundance of chordate over microbial reads suggests no addition of experimental error through heparinase I treatment. We report a novel and practical approach to heparinase treatment for human plasma collected and frozen in sodium heparin for several years. This is an effective demonstration of utilizing heparin plasma for NGS and downstream transcriptomic research, which could then be integrated with metabolomics from the same samples, maximizing efficiency and minimizing blood draws.


Introduction
Advancements in technology have led to a revolution in metabolomics, transcriptomics, and proteomics, all now possible utilizing small amounts of plasma.A significant challenge is the choice of anticoagulant to derive the plasma, with ethylenediaminetetraacetic acid (EDTA), heparin, fluorides, and anticoagulant citrate dextrose (ACD) all widely available.Plasma drawn and stored in heparin is most similar to serum when utilized for metabolomics as compared to other anticoagulants such as ACD, EDTA, and fluorides [1].On the other hand, heparin interferes with reverse transcriptase and restricts downstream analysis, such as qPCR and sequencing [2,3].
Cell-free RNAs (cfRNAs) are increasingly promising analytes in both cancer-and noncancer-related research [4][5][6][7].Successful removal of heparin and subsequent qPCR have been highly challenging, limiting use for the study of cfRNAs.Though heparinase enzymes can degrade heparin and thus allow downstream analysis, RNA often becomes denatured during incubation.RNAse inhibitors, MgCl 2, and different buffers could potentially prevent the degradation of cfRNAs.Successful heparinase treatment along with qRT-PCR and microRNA profiling in different body fluids and disease conditions has been previously demonstrated [3,[8][9][10].However, to the best of our knowledge, there has not yet been a study that has examined heparinase treatment on cell-free plasma RNA followed by library preparation and next-generation sequencing (NGS) in humans.
In the present study, we aimed to develop a simple, time-efficient, and cost-effective heparinase protocol, followed by library preparation and sequencing of plasma drawn and stored in heparin at −80 • C for several years from patients with chronic hepatitis C virus (HCV) infection.

Experimental Design
We have used human plasma from patients infected with HCV for different experiments in this study.Patients with chronic HCV infection were assessed and enrolled (NCT02400216) at the National Institutes of Health (NIH) Clinical Center [11].The study was approved by The National Institute of Diabetes, Digestive and Kidney Diseases, the National Institute of Arthritis and Musculoskeletal Diseases, and the Institutional Review Board at NIH.

Materials Human Plasma
Seven to eight mL of peripheral blood samples was obtained from an antecubital vein and drawn into CPT™ sodium heparin tubes (BD Biosciences, Ann Arbor, MI, USA, Cat# 362753) and spun down at 1600 rpm for 10 min at 4 • C for plasma.Then, 1 mL of plasma was aliquoted in multiple Eppendorf tubes, and all the samples were stored at −80 • C until analysis was complete.Plasma samples collected in sodium EDTA as well as in sodium heparin from anonymized healthy donors were obtained from the NIH Clinical Center Blood Bank.

cDNA Synthesis, Library Preparation, and Sequencing
After 1 h of incubation at 37 • C, samples treated with Bacteroides heparinase I were taken for cDNA synthesis, followed by library preparation according to the manufacturer's recommendations (NEB, Cat# E7770, Ipswich, MA, USA).Then, 4 µL of Bacteroides heparinase-I-treated sample was mixed with 4.6 µL of NEBNext First Strand Synthesis Reaction Buffer (5X) and 1.15 µL of NEBNext Random Primers followed by 15 min incubation at 94 • C in a pre-heated thermal cycler (BIO-RAD, Hercules, CA, USA, C1000 Touch Thermal Cycler) and then immediately placed on ice.After that, 2 µL of NEBNext First Strand Synthesis Enzyme Mix and 8 µL of nuclease-free water (supplied with the kit, NEB, Cat# E7770, Ipswich, MA, USA) were added to each sample and placed on the pre-heated thermal cycler for following cycles: 25 • C for 10 min, 42 • C for 50 min, 70 • C for 15 min, and 4 • C hold for 10 min.For second-strand cDNA synthesis, a master mix was made using NEBNext Second Strand Synthesis Reaction Buffer (10X) (8 µL), NEBNext Second Strand Synthesis Enzyme Mix (4 µL), and nuclease-free water (48 µL) for each sample.After adding the second-strand master mix to the sample, the mixture was well mixed with a pipette and incubated in the thermal cycler at 16 • C for 1 h.Then, the cDNAs were purified using SPRIselect beads (Beckman Coulter; Cat# B23317, Brea, CA, USA) and used for library preparation.Each library was mixed with 25 µL of NEBNext Ultra II Q5 Master Mix (provided with the kit, NEB, Cat# E7770, Ipswich, MA, USA) and was tagged using NEBNext Multiplex Oligos for Illumina (NEB, Cat# E6609) for PCR (Table 2) in the pre-heated thermal cycler.All the RNA libraries were checked using a High Sensitivity D5000 ScreenTape ® kit (Agilent, Cat#5067-5592, Santa Clara, CA, USA) in TapeStation (Agilent 4200 TapeSta-tion System) for quality purposes and quantified using Quant-iT™ PicoGreen™ dsDNA Assay Kits (Thermo Fisher Scientific, Cat# P7581, Eugene, OR, USA) in a TBS-380 Mini-Fluorometer (Turner BioSystems, Sunnyvale, CA, USA).Libraries were sequenced on a NovaSeq6000 System (Illumina, San Diego, CA, USA) for 50 base pair (bp) paired-end reads from The National Heart, Lung, and Blood Institute (NHLBI, Bethesda, MD, USA) DNA Sequencing and Genomics Core.

Expected Results
Current Bacteroides heparinase I treatment is well-suited for a wide range of cfRNA concentrations.
In this study, the average concentration of extracted cfRNA was 78.09 ± 10.66 ng/mL of heparin plasma with a range of 10.59-164.04 ng/mL of heparin plasma.The average amount of RNA used for Bacteroides heparinase I treatment was approximately 52 ± 7.11 ng (range is 7.06-111.76ng) (Figure 1).Bacteroides heparinase-I-treated libraries were suitable for metagenomic analysis.

Discussion
Plasma cfRNAs are considered non-invasive markers in different diseases and a potential source for both human and microbial signatures in blood [16][17][18].Many previous studies analyzing cfRNAs used EDTA plasma, as heparin is a known inhibitor of qPCRbased downstream analysis.As a result, blood is usually collected into various types of tubes, increasing the risk of sampling error, contamination, burden of work, and cost.In the present study, we have designed a cost-effective and straightforward protocol for heparinase treatment (using Bacteroides heparinase I enzyme) of plasma stored in sodium heparin for many years at −80 °C followed by next-generation sequencing (NGS) to show feasibility for qPCR-based downstream analysis.
Instability and low concentrations are two significant challenges for the study of heparinized plasma cfRNAs, making it harder to incubate samples for a long time with heparinase enzymes [2].Plasma cfRNA concentrations depend on the plasma volume, type of subjects, and the extraction procedures.As reported by other studies, a 1 h incubation with enzyme mixtures [8,19] helps in the removal of heparin and also allows successful

Discussion
Plasma cfRNAs are considered non-invasive markers in different diseases and a potential source for both human and microbial signatures in blood [16][17][18].Many previous studies analyzing cfRNAs used EDTA plasma, as heparin is a known inhibitor of qPCR-based downstream analysis.As a result, blood is usually collected into various types of tubes, increasing the risk of sampling error, contamination, burden of work, and cost.In the present study, we have designed a cost-effective and straightforward protocol for heparinase treatment (using Bacteroides heparinase I enzyme) of plasma stored in sodium heparin for many years at −80 • C followed by next-generation sequencing (NGS) to show feasibility for qPCR-based downstream analysis.
Instability and low concentrations are two significant challenges for the study of heparinized plasma cfRNAs, making it harder to incubate samples for a long time with heparinase enzymes [2].Plasma cfRNA concentrations depend on the plasma volume, type of subjects, and the extraction procedures.As reported by other studies, a 1 h incubation with enzyme mixtures [8,19] helps in the removal of heparin and also allows successful library preparation, even with meager quantities of starting cfRNAs (~7 ng).Use of the buffer from the same cDNA synthesis and library preparation kit makes this method of heparinase treatment far more time-and cost-efficient than others [8,19].Furthermore, many of the previously published heparinase treatment studies were performed with fresh plasma, whereas the described approach is suitable for frozen plasma stored for at least up to 6 years at −80 • C.
Though there are differences in the read count and reproducibility in NGS data, it has been well established that a far greater number of plasma cfRNAs align with the human genome compared to microbial genomes [7].This finding is consistent with the present study.It has been difficult to find studies that deal with plasma stored in sodium heparin, which were then utilized for NGS to analyze both human and microbial sequences, making the described protocol a novel approach for heparinase treatment and subsequent NGS.
There are a few limitations of the current study.To minimize the degradation of cfRNAs, no experiments were performed to investigate the heparin concentration in the samples before and after heparinase treatment.No comparison was performed between EDTA plasma, non-treated heparin plasma, and treated heparin plasma in the present study in terms of extracted cfRNA concentrations and NGS.As a result, the effect of different anticoagulants on plasma cfRNA populations is not addressed in this manuscript.Multiple library samples stored in EDTA and heparin (treated and non-treated with heparinase) plasma from healthy blood donors were analyzed through the 2100 expert_High Sensitivity DNA Assay kit to confirm successful library preparation (Figure 3) after heparinase treatment.As our goal is to establish an authentic approach for heparinase treatment only, we do not present any analysis of microbial species in this paper.Clinical and epidemiological studies can continue over the span of several years, with multiple time points and interventions.It would be efficient and effective if there were one suitable anticoagulant for preserving plasma to perform metabolomics and transcriptomics.Here we present a simple and authentic approach to removing heparin from frozen, heparinized plasma stored for several years and subsequent use for qPCR-based downstream analysis.

Conclusions
The current study is a novel and practical approach to heparinase treatment for human plasma collected and frozen in sodium heparin for several years.The protocol is an effective demonstration of the removal of heparin in plasma as a barrier to transcriptomic research.

Conclusions
The current study is a novel and practical approach to heparinase treatment for human plasma collected and frozen in sodium heparin for several years.The protocol is an effective demonstration of the removal of heparin in plasma as a barrier to transcriptomic research.Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.
Data Availability Statement: Any data not published in this manuscript is available upon reasonable request from the corresponding author.

10 Figure 1 .
Figure 1.cfRNA concentrations in the HCV heparin plasma.cfRNA concentrations varied from sample to sample (A).As 4 µL of water-suspended cfRNAs was directly used (without correction of concentration) for heparinase treatment, starting cfRNA amounts were different for these samples (B).

Figure 1 .
Figure 1.cfRNA concentrations in the HCV heparin plasma.cfRNA concentrations varied from sample to sample (A).As 4 µL of water-suspended cfRNAs was directly used (without correction of concentration) for heparinase treatment, starting cfRNA amounts were different for these samples (B).

Figure 2 .
Figure 2. cfRNA libraries from heparinized plasma of HCV patients.Library quality control report from High Sensitivity D5000 ScreenTape ® (A,B).Green and Purple color lines in figure B indicate lowest and highest ladder respectively present during TapeStation analysis.Library sizes for 17 HCV patients (C).Percentages of multiple subtypes of reads found in the HCV patient samples (D).

Figure 2 .
Figure 2. cfRNA libraries from heparinized plasma of HCV patients.Library quality control report from High Sensitivity D5000 ScreenTape ® (A,B).Green and Purple color lines in figure B indicate lowest and highest ladder respectively present during TapeStation analysis.Library sizes for 17 HCV patients (C).Percentages of multiple subtypes of reads found in the HCV patient samples (D).

Figure 3 .
Figure 3. cfRNA libraries from healthy blood donors.Panel (A) and (B) represent library from Bacteroides heparinase-I-treated and non-treated heparin plasma, respectively, and (C) represents libraries from EDTA plasma.Experiments were carried out in multiple batches in multiple days.A cfRNA amount of ~8-50 ng per 1 mL of plasma was used for the upper experiments.Green and Purple color lines indicate lowest and highest ladder respectively present in bioanalyzer.

Figure 3 .
Figure 3. cfRNA libraries from healthy blood donors.Panel (A) and (B) represent library from Bacteroides heparinase-I-treated and non-treated heparin plasma, respectively, and (C) represents libraries from EDTA plasma.Experiments were carried out in multiple batches in multiple days.A cfRNA amount of ~8-50 ng per 1 mL of plasma was used for the upper experiments.Green and Purple color lines indicate lowest and highest ladder respectively present in bioanalyzer.

Author Contributions:
All authors made a substantial contribution to this work.Individual contributions are listed as follows: R.A.-Conception and design of the work; acquisition, analysis, interpretation of data; drafted the initial work and reviewed critically for important intellectual content; N.M.-Acquisition of data and reviewed critically for important intellectual content; J.B.L.-Analysis and interpretation of data; M.C.-critical review and editing for important intellectual content; J.A.H.-Acquisition of data; R.O.A.-Acquisition of data; C.K.-Acquisition of data; E.B.L.-Acquisition of data; O.E.-Acquisition of data; T.H.-conception and design of the work; the acquisition, analysis, interpretation of data; drafting the initial work and critical review for important intellectual content.All authors have read and agreed to the published version of the manuscript.Funding: This research was funded by intramural program of NIDDK, grant number [1ZIADK054514] (T.H.), and the Clinical Center, NIH.Institutional Review Board Statement: The study was conducted in accordance with the Declaration of Helsinki, and approved by the Institutional Review of NIH (NCT02400216, March, 2015).

Table 2 .
Conditions for PCR enrichment during library preparation.